We first synthesized and characterized unnatural (+)-naloxone in 1978 as an opioid receptor inert research tool useful for detecting opioid receptor mediated effects when used in conjunction with the life-saving narcotic antagonist (-)-naloxone. In recent years, we have shown that (+)-naloxone and the related (+)-naltrexone are functional antagonists of toll-like receptor 4 (TLR4) and that selective functional antagonism of TLR4 by (+)-naloxone can result in reversal of neuropathic pain as well as potentiation of opioid analgesia. TLR4 is known to be involved in many physiological functions via mediation of glial activation including, neuropathic pain, compromised acute opioid analgesia, and unwanted opioid side effects (tolerance, dependence, and reward) and inflammation. Attenuation of glial activation has been shown to both to alleviate exaggerated pain states induced by experimental pain models and to reduce the development of opioid tolerance. In order to gain further insight into the interaction of (+)- naltrexone with its binding pocket in the myeloid differentiation protein 2 (MD-2) and resulting blockade of the TLR4 signal cascade, we studied the interaction of MD-2 and (+)-naltrexone and (+)-naloxone with in silico and in vitro analyses. Our results showed that these (+)-isomers interacted with the hydrophobic portions of MD-2 and that increasing the lipophilicity of the nitrogen substituent generally increased the TLR4 antagonism in vitro. The most active compound in the series was found when the N-cyclopropylmethyl substituent of (+)-naltrexone was replaced by the N-phenethyl group. The present study provides valuable insight into structure-activity relationships in this series for development of the next generation TLR4 antagonists.